U.S. patent application number 14/644640 was filed with the patent office on 2015-09-17 for ugt8 mini-promoters.
The applicant listed for this patent is The University of British Columbia. Invention is credited to Vikramjit Chopra, Cletus D'Souza, Charles de Leeuw, Daniel Goldowitz, Robert A. Holt, Steven J. Jones, Elodie Portales-Casamar, Elizabeth M. Simpson, Wyeth W. Wasserman.
Application Number | 20150259691 14/644640 |
Document ID | / |
Family ID | 54068252 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150259691 |
Kind Code |
A1 |
Simpson; Elizabeth M. ; et
al. |
September 17, 2015 |
UGT8 MINI-PROMOTERS
Abstract
Isolated polynucleotides comprising a UGT8 mini-promoters are
provided. The mini-promoter may be operably linked to an
expressible sequence, e.g. reporter genes, genes encoding a
polypeptide of interest, regulatory RNA sequences such as miRNA,
siRNA, anti-sense RNA, etc., and the like. In some embodiments a
cell comprising a stable integrant of an expression vector is
provided, which may be integrated in the genome of the cell. The
promoter may also be provided in a vector, for example in
combination with an expressible sequence. The polynucleotides find
use in a method of expressing a sequence of interest, e.g. for
identifying or labeling cells, monitoring or tracking the
expression of cells, gene therapy, etc.
Inventors: |
Simpson; Elizabeth M.;
(Vancouver, CA) ; D'Souza; Cletus; (Vancouver,
CA) ; de Leeuw; Charles; (Seattle, WA) ;
Goldowitz; Daniel; (Port Moody, CA) ; Holt; Robert
A.; (North Vancouver, CA) ; Portales-Casamar;
Elodie; (Vancouver, CA) ; Chopra; Vikramjit;
(Vancouver, CA) ; Wasserman; Wyeth W.; (Vancouver,
CA) ; Jones; Steven J.; (Vancouver, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of British Columbia |
Vancouver |
|
CA |
|
|
Family ID: |
54068252 |
Appl. No.: |
14/644640 |
Filed: |
March 11, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61951378 |
Mar 11, 2014 |
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Current U.S.
Class: |
514/44R ;
435/320.1; 435/325; 435/455; 536/24.1 |
Current CPC
Class: |
C12N 9/1051 20130101;
C12N 15/85 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12N 9/10 20060101 C12N009/10 |
Claims
1. An isolated polynucleotide comprising a UGT8 mini-promoter
wherein the UGT8 mini-promoter comprises at least one UGT8
regulatory element with substantial similarity to SEQ ID NO: 5, 6,
7, 8, 9 or 10; operably joined to a UGT8 basal promoter with
substantial similarity to SEQ ID NO: 3 or 4 through a non-native
spacing between the UGT8 regulatory element and the UGT8 basal
promoter.
2. The isolated polynucleotide of claim 1, comprising 2 or more
UGT8 regulatory elements.
3. The isolated polynucleotide of claim 1, comprising 3 or more
UGT8 regulatory elements.
4. The polynucleotide of claim 1, comprising a UGT8 mini-promoter
with substantial similarity to SEQ ID NO: 1.
5. The polynucleotide of claim 1, comprising a UGT8 mini-promoter
with substantial similarity to SEQ ID NO: 2.
6. The isolated polynucleotide of claim 1, operably linked to an
expressible sequence.
7. A vector comprising the isolated polynucleotide of claim 1.
8. An isolated cell comprising the vector of claim 7.
9. The cell of claim 8, wherein the vector is stably integrated
into the genome of the cell.
10. The cell of claim 8, wherein the cell is a stem cell, a retinal
cell, a brain or a glial cell.
11. A method of expressing a sequence of interest, the method
comprising operably linking the sequence of interest to the
polynucleotide of claim 1; and introducing into a cell permissive
for expression from the UGT8 mini-promoter.
12. The method of claim 11, wherein the cells are Muller glia cells
or a progenitor thereof.
13. The method of claim 11, wherein the cells are oligodendrocytes
or progenitors thereof.
14. A method of treatment of a subject having a disease or
condition of the eye or brain, the method comprising administering
to the subject a therapeutically effective dose of a composition
comprising a polynucleotide of claim 1.
15. An isolated polynucleotide comprising a UGT8 mini-promoter
wherein the UGT8 mini-promoter comprises at least one UGT8
regulatory element with at least 95% sequence identity to SEQ ID
NO: 6, 7, 8, 9 or 10 operably joined to an UGT8 basal promoter with
at least 95% sequence identity to SEQ ID NO: 4 or 5, wherein the
spacing between the UGT8 regulatory element and the UGT8 basal
promoter is not more than 500 nucleotides (nt).
16. The polynucleotide of claim 15, wherein the UGT8 mini-promoter
has at least 95% sequence identity to SEQ ID NO: 1.
17. The polynucleotide of claim 15 wherein the UGT8 mini-promoter
has at least 95% sequence identity to SEQ ID NO: 2.
18. The isolated polynucleotide of claim 11, operably linked to an
expressible sequence.
Description
FIELD OF THE INVENTION
[0001] The invention relates to gene promoters and regulatory
elements. More specifically, the invention relates to novel UGT8
promoter compositions and related methods.
BACKGROUND
[0002] The UGT8 gene encodes the UDP glycosyltransferase 8A,
catalyzing the transfer of galactose to ceramide, a key enzymatic
step in the biosynthesis of galactocerebrosides, which are abundant
sphingolipids of the myelin membrane of the CNS and PNS. This
biological function implicates UGT8 as an important gene in
oligodendrocyte function. The expression pattern on the Allen Brain
Atlas data indicates that this gene is expressed along axonal
tracks--this in situ hybridization data is consistent with
expression in oligodendrocytes. Schulte and Stoffel (1993)
demonstrated that the expression is restricted to
oligodendrocyte-containing layers of the cerebrum and cerebellum,
showing the same expression distribution in brain as myelin basic
protein.
[0003] There is a need for characterized human UGT8 promoters for
gene expression, for instance in human gene therapy applications.
It is in particular useful to identify small promoter elements that
are sufficient to drive expression in certain cell types, for
instance retinal cells. Such small promoter elements, or
"mini-promoters" are particularly useful in certain applications,
for instance they are more amenable to insertion into viral vectors
used in gene therapy applications.
SUMMARY OF THE INVENTION
[0004] The present invention provides novel nucleic acid sequence
compositions and methods relating to minimal human UGT8 promoters.
The invention is based in part on the surprising discovery that
certain minimal UGT8 promoter elements are capable of expressing in
specific cell types, for instance in cells of the brain or eye.
[0005] In one embodiment of the invention, there is provided an
isolated nucleic acid fragment comprising an UGT8 mini-promoter,
wherein the UGT8 mini-promoter comprises one or more UGT8
regulatory elements operably linked in a non-native conformation to
a UGT8 basal promoter. The UGT8 mini-promoter may have a nucleic
acid sequence which is substantially similar in sequence and
function to SEQ ID NO: 1 or 2. The UGT8 basal promoter may have a
nucleic acid sequence which is substantially similar in sequence
and function to SEQ ID NO: 3 or 4. The UGT8 regulatory element may
have a nucleic acid sequence which is substantially similar in
sequence and function to one or more of SEQ ID NO: 5-10, e.g.
comprising one, two, three, four or five of the regulatory elements
set forth in the provided sequences. The UGT8 mini-promoters may
further be operably linked to an expressible sequence, e.g.
reporter genes, genes encoding a polypeptide of interest,
regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA,
etc., and the like. Reporter gene sequences include, for example
luciferase, beta-galactosidase, green fluorescent protein, enhanced
green fluorescent protein, and the like as known in the art. The
expressible sequence may encode a protein of interest, for example
a therapeutic protein, receptor, antibody, growth factor, and the
like. The expressible sequence may encode an RNA interference
molecule.
[0006] In one embodiment, there is provided an expression vector
comprising an UGT8 mini-promoter, wherein the UGT8 mini-promoter
comprises one or more UGT8 regulatory elements operably linked in a
non-native conformation to an UGT8 basal promoter. The UGT8
mini-promoter may have a nucleic acid sequence which is
substantially similar in sequence and function to SEQ ID NO: 1 or
2. The UGT8 basal promoter may have a nucleic acid sequence which
is substantially similar in sequence and function to one or more of
SEQ ID NO: 3 or 4. The one or more UGT8 regulatory elements may
have a nucleic acid sequence which is substantially similar in
sequence and function to SEQ ID NO: 5-10. The UGT8 mini-promoter
may further be operably linked to an expressible sequence, e.g.
reporter genes, genes encoding a polypeptide of interest,
regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA,
etc., and the like. Reporter gene sequences include, for example
luciferase, beta-galactosidase, green fluorescent protein, enhanced
green fluorescent protein, and the like as known in the art. The
expressible sequence may encode a protein of interest, for example
a therapeutic protein, receptor, antibody, growth factor, and the
like. The expressible sequence may encode an RNA interference
molecule. The expression vector may further comprise a genomic
targeting sequence. The genomic targeting sequence may be HPRT.
[0007] In one embodiment, there is provided a method for expressing
a gene, protein, RNA interference molecule or the like in a cell,
the method comprising introducing into the cell an expression
vector comprising an UGT8 mini-promoter element, wherein the UGT8
mini-promoter element comprises one or more UGT8 regulatory
elements operably linked in a non-native conformation to an UGT8
basal promoter element. Cells of interest include, without
limitation, cells of the peripheral or central nervous system and
progenitors thereof, e.g. embryonic stem cells, neural stem cells,
neurons, glial cells, astrocytes, microgial cells, etc; and/or
cells in the eye and progenitors thereof, e.g. retinal cells,
Muller glia cells, etc. The UGT8 mini-promoter may have a nucleic
acid sequence which is substantially similar in sequence and
function to SEQ ID NO: 1 or 2. The UGT8 basal promoter may have a
nucleic acid sequence which is substantially similar in sequence
and function to SEQ ID NO: 3 or 4. The UGT8 regulatory element may
have a nucleic acid sequence which is substantially similar in
sequence and function to one or more of SEQ ID NO: 5-10. The UGT8
mini-promoter may further be operably linked to an expressible
sequence, e.g. reporter genes, genes encoding a polypeptide of
interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense
RNA, etc., and the like. Reporter gene sequences include, for
example luciferase, beta-galactosidase, green fluorescent protein,
enhanced green fluorescent protein, and the like as known in the
art. The expressible sequence may encode a protein of interest, for
example a therapeutic protein, receptor, antibody, growth factor,
and the like. The expressible sequence may encode an RNA
interference molecule. The expression vector may thus further
comprise a genomic targeting sequence. The genomic targeting
sequence may be HPRT.
[0008] In one embodiment of the invention, there is provided a
method for identifying or labeling a cell, the method comprising
introducing into the cell an expression vector comprising an UGT8
mini-promoter element, wherein the UGT8 mini-promoter element
comprises one or more UGT8 regulatory element operably linked in a
non-native conformation to a UGT8 basal promoter element, and
wherein the expressible sequence comprises a reporter gene. The
UGT8 mini-promoter element may have a nucleic acid sequence
substantially similar in sequence and function to SEQ ID NO: 1 or
2. The UGT8 basal promoter element may have a nucleic acid sequence
substantially similar in sequence and function to SEQ ID NO: 3 or
4. The UGT8 regulatory element may have a nucleic acid sequence
substantially similar in sequence and function to one or more of
SEQ ID NO: 5-10. In some embodiments, the cell is a peripheral or
central nervous system cell or progenitors thereof, including,
without limitation, embryonic stem cells, neural stem cells, glial
cells, astrocytes, neurons and the like, and/or cells in the eye
and progenitors thereof, e.g. retinal cells, retinal Muller glia,
etc. Reporter gene sequences include, for example luciferase,
beta-galactosidase, green fluorescent protein, enhanced green
fluorescent protein, and the like as known in the art. The
expressible sequence may encode a protein of interest, for example
a therapeutic protein, receptor, antibody, growth factor, RNA
interference molecule and the like.
[0009] In one embodiment of the invention, there is provided a
method for monitoring or tracking the development or maturation of
a cell, the method comprising: 1) introducing into the cell an
expression vector comprising an UGT8 mini-promoter element operably
linked to an expressible sequence, wherein the UGT8 mini-promoter
element comprises one or more UGT8 regulatory elements operably
linked in a non-native conformation to an UGT8 basal promoter
element, and wherein the expressible sequence comprises a reporter
gene; and 2) detecting the expression of the reporter gene in the
cell of in progeny of the cell as a means of determining the
lineage, identity or developmental state of the cell or cell
progeny. The UGT8 mini-promoter element may have a nucleic acid
sequence substantially similar in sequence and function to SEQ ID
NO: 1 or 2. The UGT8 basal promoter element may have a nucleic acid
sequence substantially similar in sequence and function to SEQ ID
NO: 3 or 4. The UGT8 regulatory element may have a nucleic acid
sequence substantially similar in sequence and function to one or
more of SEQ ID NO: 5-10. In some embodiments, the cell is a
peripheral or central nervous system cell or progenitors thereof,
including, without limitation, embryonic stem cells, neural stem
cells, glial cells, neurons and the like. In some embodiments, the
cell is an eye cell or progenitor thereof, including without
limitation a retinal cell, a retinal Muller glial cell, and the
like.
[0010] In certain embodiments of the invention, there is thus
provided a method of treatment of a subject having a disease or
condition of the eye, the method comprising administering to the
subject a therapeutically effective dose of a composition
comprising an UGT8 mini-promoter element, wherein the UGT8
mini-promoter element comprises one or more UGT8 regulatory
elements operably linked in a non-native conformation to an UGT8
basal promoter element. The UGT8 mini-promoter element may have a
nucleic acid sequence substantially similar in sequence and
function to SEQ ID NO: 1 or 2. The UGT8 basal promoter element may
have a nucleic acid sequence substantially similar in sequence and
function to SEQ ID NO: 3 or 4. The UGT8 regulatory element may have
a nucleic acid sequence substantially similar in sequence and
function to SEQ ID NO: 5-10. The disease or condition may be chosen
from: retinal diseases, retinal degeneration, retinal damage,
blindness, macular degeneration, retinitis pigmentosa, inherited
retinal genetic diseases, diabetic retinopathy, cone rod dystrophy,
hypertensive/diabetic retinopathy. The therapeutic or beneficial
compound may be a light-sensitive compound, for instance rhodopsin,
channel rhodopsin, etc.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0012] FIG. 1--DNA expression vector (pEMS1313) into which UGT8
promoter elements were inserted for expression studies. The UGT8
promoter with a nucleic acid sequence corresponding to SEQ ID NO: 1
or 2 was inserted into the multiple cloning site (MCS) of the
pEMS1313 vector such that it became operably linked to the lac Z
reporter gene. The final construct, called Ple240 (containing SEQ
ID NO: 1) or Ple267 (containing SEQ ID NO: 2), also contained the
HPRT genomic targeting sequence, an ampicillin resistance gene
(AmpR) for screening, and a transcriptional termination sequence
(SV40 polyA), as well as other elements necessary for vector
replication and gene expression.
[0013] FIG. 2--Ple240 (UGT8 RRs) express in regions containing glia
in the mouse brain and eye. The Ple240 promoter driving
beta-galactosidase expression was constructed and knocked-in as a
single-copy at the mouse Hprt locus for reproducible expression.
Adult mice were harvested via perfusion and stained overnight for
lacZ activity (blue), indicative of promoter activity. A, a coronal
and sagittal slice of mouse brain shows expression in all white
matter regions (associated with oligodendroglia), in cortex layer
IV (a known region of oligodendrocytes), and in the olfactory
globeruli (an area with extensive myelination). B, similarly there
is expression along the nerve bundles exiting the spinal cord,
which are consistent with Schwann cells (the oligodendroglia
outside of the CNS). C, in the mouse eye we see the long processes
spanning the entire retina with nuclei in the inner nuclear layer
(indicative of Muller glia cells; the supportive glia in the
retina).
[0014] FIG. 3--Ple267 (UGT8 RRs) express in regions containing glia
in the mouse brain and spinal cord. The Ple267 promoter driving
icre expression was constructed and ssAAV9 virus generated
(vEMS40). P0 mice were injected intravenously with virions and mice
were harvested at P56 via perfusion and stained overnight for lacZ
activity (blue), indicative of promoter activity. A, a sagittal
cryosection of mouse brain shows expression in the cortex (Ctx) and
corpus callosum (CC), with "puffy" cell bodies indicative of
oligodendrocytes and consistent with their known location. B, in a
sagittal 1-mm brain section, areas of white matter are often
stained positive. C, similarly there is expression along the nerve
bundles exiting the spinal cord, which are consistent with Schwann
cells (the oligodendroglia outside of the CNS).
DETAILED DESCRIPTION
[0015] The compositions of the present invention include novel
polynucleotides comprising UGT8 promoter elements (also referred to
herein as UGT8 mini-promoters) as well as novel expression vectors
comprising said UGT8 promoter elements (or mini-promoters). The
present invention also includes various methods utilizing these
novel UGT8 promoter (or mini-promoter) elements or expression
vectors.
[0016] The term `UGT8` refers to the gene which encodes the UGT8
protein, also referred to UDP glycosyltransferase 8. The human
homolog of UGT8 is encoded by the human gene identified as
EntrezGene #7368 and is located at chromosomal location 4q26. The
protein encoded by human UGT8 has the Protein Accession # Q16880.2,
however other protein accession numbers may also be assigned to
this protein. UGT8 may also include other isoforms and/or splice
variants. Other mammalian UGT8 homologs may include but are not
limited to: Rattus norvegicus (EntrezGene #50555), Mus musculus
(EntrezGene #22239).
[0017] The term `promoter` refers to the regulatory DNA region
which controls transcription or expression of a gene and which can
be located adjacent to or overlapping a nucleotide or region of
nucleotides at which RNA transcription is initiated. A promoter
contains specific DNA sequences which bind protein factors, often
referred to as transcription factors, which facilitate binding of
RNA polymerase to the DNA leading to gene transcription. A `basal
promoter`, also referred to as a `core promoter`, usually means a
promoter which contains all the basic necessary elements to promote
transcriptional expression of an operably linked polynucleotide.
Eukaryotic basal promoters typically, though not necessarily,
contain a TATA-box and/or a CAAT box. A `UGT8 basal promoter`, in
the context of the present invention and as used herein, is a
nucleic acid compound having a sequence with at least 65%, at least
70%, at least 80%, at least 85%, at least 90%, at least 95%, or at
least 99% similarity to SEQ ID NO: 3 or 4.
[0018] A promoter may also include `regulatory elements` that
influence the expression or transcription by the promoter. Such
regulatory elements encode specific DNA sequences which bind other
factors, which may include but are not limited to enhancers,
silencers, insulators, and/or boundary elements. A `UGT8 regulatory
element`, in the context of the present invention and as used
herein, is a nucleic acid compound having a sequence with at least
65%, at least 70%, at least 80%, at least 85%, at least 90%, at
least 95%, or at least 99% similarity to any one of SEQ ID NO:
5-10. The present invention provides, in certain embodiments as
described herein, different promoters of the UGT8 gene. In some
embodiments, the UGT8 promoter comprises one or more UGT8
regulatory elements operably linked to a UGT8 basal promoter.
[0019] The term `operably linked`, in the context of the present
invention, means joined in such a fashion as to work together to
allow transcription. In some embodiments of the invention, two
polynucleotide sequences may be operably linked by being directly
linked via a nucleotide bond. In this fashion, the two operably
linked elements contain no intervening sequences and in being
joined are able to direct transcription of an expression sequence.
In other embodiments of the invention, two elements may be operably
linked by an intervening compound, for instance a polynucleotide
sequence of variable length. In such a fashion, the operably linked
elements, although not directly juxtaposed, are still able to
direct transcription of an expression sequence. Thus, according to
some embodiments of the invention, one or more promoter elements
may be operably linked to each other, and additionally be operably
linked to a downstream expression sequence, such that the linked
promoter elements are able to direct expression of the downstream
expression sequence.
[0020] The term `mini-promoter` refers to a promoter in which
certain promoter elements are selected from an endogenous full
length promoter for a gene, usually in such a fashion as to reduce
the overall size of the promoter compared to the native sequence.
For example, after identification of critical promoter elements,
using one or more of various techniques, the native sequences that
intervene between identified elements may be partially or
completely removed. Other non-native sequences may optionally be
inserted between the identified promoter elements. Promoter
sequences such as enhancer elements may have an orientation that is
different from the native orientation--for example, a promoter
element may be inverted, or reversed, from its native orientation.
Alternatively, selecting a minimal basal promoter that is
sufficient to drive expression in particular cells or tissues may
also be desirable. Since promoter elements that impact expression
patterns are known to be distributed over varying distances of the
proximal and/or distal endogenous promoter, it is a non-trivial
task to identify a mini-promoter comprising a minimal basal
promoter and optional regulatory regions that will adequately
express in the desired cell or tissue types. A mini-promoter may
provide certain advantages over native promoter conformations. For
example, the smaller size of the mini-promoter may allow easier
genetic manipulation, for example in the design and/or construction
of expression vectors or other recombinant DNA constructs. In
addition, the smaller size may allow easier insertion of DNA
constructs into host cells and/or genomes, for example via
transfection, transformation, etc. Other advantages of
mini-promoters are apparent to one of skill in the art. In some
embodiments of the invention, there are thus provided novel UGT8
mini-promoters comprising a UGT8 regulatory element operably linked
in a non-native conformation to a UGT8 basal promoter. In general
the spacing between the UGT8 regulatory element and the UGT8 basal
promoter is not more than about 15 KB, generally not more than
about 10 KB, usually not more than about 1 KB, more often not more
than about 500 nt, and may be not more than about 100 nt, down to a
direct joining of the two sequences. In other embodiments, there is
provided a minimal UGT8 basal promoter.
[0021] The term `expressible sequence` refers to a polynucleotide
composition which is operably linked to a promoter element such
that the promoter element is able to cause transcriptional
expression of the expression sequence. An expressible sequence is
typically linked downstream, on the 3'-end of the promoter
element(s) in order to achieve transcriptional expression. The
result of this transcriptional expression is the production of an
RNA macromolecule. The expressed RNA molecule may encode a protein
and may thus be subsequently translated by the appropriate cellular
machinery to produce a polypeptide protein molecule. In some
embodiments of the invention, the expression sequence may encode a
reporter protein. Alternately, the RNA molecule may be an
antisense, RNAi or other non-coding RNA molecule, which may be
capable of modulating the expression of specific genes in a cell,
as is known in the art.
[0022] The term `RNA` as used in the present invention includes
full-length RNA molecules, which may be coding or non-coding
sequences, fragments, and derivatives thereof. For example, a
full-length RNA may initially encompass up to about 20 Kb or more
of sequence, and frequently will be processed by splicing to
generate a small mature RNA. Fragments, RNAi, miRNA and anti-sense
molecules may be smaller, usually at least about 18 nt. in length,
at least about 20 nt in length, at least about 25 nt. in length,
and may be up to about 50 nt. in length, up to about 100 nt in
length, or more. RNA may be single stranded, double stranded,
synthetic, isolated, partially isolated, essentially pure or
recombinant. RNA compounds may be naturally occurring, or they may
be altered such that they differ from naturally occurring RNA
compounds. Alterations may include addition, deletion, substitution
or modification of existing nucleotides. Such nucleotides may be
either naturally occurring, or non-naturally occurring nucleotides.
Alterations may also involve addition or insertion of
non-nucleotide material, for instance at the end or ends of an
existing RNA compound, or at a site that is internal to the RNA
(ie. between two or more nucleotides).
[0023] The term `nucleic acid` as used herein includes any nucleic
acid, and may be a deoxyribonucleotide or ribonucleotide polymer in
either single or double-stranded form. A `polynucleotide` or
`nucleotide polymer` as used herein may include synthetic or mixed
polymers of nucleic acids, both sense and antisense strands, and
may be chemically or biochemically modified or may contain
non-natural or derivatized nucleotide bases, as will be readily
appreciated by those skilled in the art. Such modifications
include, for example, labels, methylation, substitution of one or
more of the naturally occurring nucleotides with an analog,
internucleotide modifications such as uncharged linkages (e.g.,
methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,
etc.), charged linkages (e. g., phosphorothioates,
phosphorodithioates, etc.), pendent moieties (e.g., polypeptides),
and modified linkages (e.g., alpha anomeric polynucleotides, etc.).
Also included are synthetic molecules that mimic polynucleotides in
their ability to bind to a designated sequence via hydrogen bonding
and other chemical interactions.
[0024] A `purine` is a heterocyclic organic compound containing
fused pyrimidine and imidazole rings, and acts as the parent
compound for purine bases, adenine (A) and guanine (G).
`Nucleotides` are generally a purine (R) or pyrimidine (Y) base
covalently linked to a pentose, usually ribose or deoxyribose,
where the sugar carries one or more phosphate groups. Nucleic acids
are generally a polymer of nucleotides joined by 3' 5'
phosphodiester linkages. As used herein `purine` is used to refer
to the purine bases, A and G, and more broadly to include the
nucleotide monomers, deoxyadenosine-5'-phosphate and
deoxyguanosine-5'-phosphate, as components of a polynucleotide
chain. A `pyrimidine` is a single-ringed, organic base that forms
nucleotide bases, such as cytosine (C), thymine (T) and uracil (U).
As used herein `pyrimidine` is used to refer to the pyrimidine
bases, C, T and U, and more broadly to include the pyrimidine
nucleotide monomers that along with purine nucleotides are the
components of a polynucleotide chain.
[0025] It is within the capability of one of skill in the art to
modify the sequence of a promoter nucleic acid sequence, e.g. the
provided basal promoter and regulatory sequences, in a manner that
does not substantially change the activity of the promoter element,
i.e. the transcription rate of an expressible sequence operably
linked to a modified promoter sequence is at least about 65% the
transcription rate of the original promoter, at least about 75% the
transcription rate of the original promoter sequence, at least
about 80%, at least about 90%, at least about 95%, at least about
99%, or more. Such modified sequences would be considered to be
`functionally similar` or to have `functional similarity` or
`substantial functional similarity` to the unmodified sequence.
Such modifications may include insertions, deletions which may be
truncation of the sequence or internal deletions, or substitutions.
The level of sequence modification to an original sequence will
determine the `sequence similarity` of the original and modified
sequences. Modification of the promoter elements of the present
invention in a fashion that does not significantly alter
transcriptional activity, as described above would result in
sequences with `substantial sequence similarity` to the original
sequence i.e. the modified sequence has a nucleic acid composition
that is at least about 65% similar to the original promoter
sequence, at least about 75% similar to the original promoter
sequence, at least about 80%, at least about 90%, at least about
95%, at least about 99%, or more similar to the original promoter
sequence. Thus, mini-promoter elements which have substantial
functional and/or sequence similarity are herein described and are
within the scope of the invention.
[0026] An `RNA interference molecule`, or `RNA interference
sequence` as defined herein, may include, but is not limited to, an
antisense RNA molecule, a microRNA molecule or a short hairpin RNA
(shRNA) molecule. Typically, RNA interference molecules are capable
of target-specific modulation of gene expression and exert their
effect either by mediating degradation of the mRNA products of the
target gene, or by preventing protein translation from the mRNA of
the target gene. The overall effect of interference with mRNA
function is modulation of expression of the product of a target
gene. This modulation can be measured in ways which are routine in
the art, for example by Northern blot assay or reverse
transcriptase PCR of mRNA expression, Western blot or ELISA assay
of protein expression, immunoprecipitation assay of protein
expression, etc.
[0027] An `antisense RNA molecule`, as used herein, is typically a
single stranded RNA compound which binds to complementary RNA
compounds, such as target mRNA molecules, and blocks translation
from the complementary RNA compounds by sterically interfering with
the normal translational machinery. Specific targeting of antisense
RNA compounds to inhibit the expression of a desired gene may
design the antisense RNA compound to have a homologous,
complementary sequence to the desired gene. Perfect homology is not
necessary for inhibition of expression. Design of gene specific
antisense RNA compounds, including nucleotide sequence selection
and additionally appropriate alterations, are known to one of skill
in the art.
[0028] The term `microRNA molecule`, `microRNA` or `miRNA`, as used
herein, refers to single-stranded RNA molecules, typically of about
21-23 nucleotides in length, which are capable of modulating gene
expression. Mature miRNA molecules are partially complementary to
one or more messenger RNA (mRNA) molecules, and their main function
is to downregulate gene expression. Without being bound by theory,
miRNAs are first transcribed as primary transcripts or pri-miRNA
with a cap and poly-A tail and processed to short, 70-nucleotide
stem-loop structures known as pre-miRNA in the cell nucleus. This
processing is performed in animals by a protein complex known as
the Microprocessor complex, consisting of the nuclease Drosha and
the double-stranded RNA binding protein Pasha. These pre-miRNAs are
then processed to mature miRNAs in the cytoplasm by interaction
with the endonuclease Dicer, which also initiates the formation of
the RNA-induced silencing complex (RISC). When Dicer cleaves the
pre-miRNA stem-loop, two complementary short RNA molecules are
formed, but only one is integrated into the RISC complex. This
strand is known as the guide strand and is selected by the
argonaute protein, the catalytically active RNase in the RISC
complex, on the basis of the stability of the 5' end. The remaining
strand, known as the anti-guide or passenger strand, is degraded as
a RISC complex substrate. After integration into the active RISC
complex, miRNAs base pair with their complementary mRNA molecules
and induce mRNA degradation by argonaute proteins, the
catalytically active members of the RISC complex. Animal miRNAs are
usually complementary to a site in the 3' UTR whereas plant miRNAs
are usually complementary to coding regions of mRNAs.
[0029] The term `short hairpin RNA` or `shRNA` refers to RNA
molecules having an RNA sequence that makes a tight hairpin turn
that can be used to silence gene expression via RNA interference.
The shRNA hairpin structure is cleaved by the cellular machinery
into siRNA, which is then bound to the RNA-induced silencing
complex (RISC). This complex binds to and cleaves mRNAs which match
the siRNA that is bound to it. shRNA is transcribed by RNA
Polymerase III whereas miRNA is transcribed by RNA Polymerase II.
Techniques for designing target specific shRNA molecules are known
in the art.
[0030] An `expression vector` is typically a nucleic acid molecule
which may be integrating or autonomous, (i.e. self-replicating),
and which contains the necessary components to achieve
transcription of an expressible sequence in a target cell, when
introduced into the target cell. Expression vectors may include
plasmids, cosmids, phage, YAC, BAC, mini-chromosomes, viruses, e.g.
retroviruses, adenovirus, lentivirus, SV-40, and the like; etc.
Many such vectors have been described in the art and are suitable
for use with the promoters of the present invention. Expression
vectors of the present invention include a promoter as described
herein, operably linked to an expressible sequence, which may also
be optionally operably linked to a transcription termination
sequence, such as a polyadenylation sequence. The expression vector
optionally contains nucleic acid elements which confer host
selectivity, elements that facilitate replication of the vector,
elements that facilitate integration of the vector into the genome
of the target cell, elements which confer properties, for example
antibiotic resistance, to the target cell which allow selection or
screening of transformed cells and the like. Techniques and methods
for design and construction of expression vectors are well known in
the art.
[0031] It may be desirable, when driving expression of an
expressible sequence with a particular promoter system to have the
expression occur in a stable and consistent manner. A factor that
has been shown to affect expression is the site of integration of
an expression vector or construct into the genome of the target
cell, sometimes called `position effects`. Such position effects
may be caused by, for example, local chromatin structure which
affects expression of sequences from that region of the genome. One
method to control for position effects when integrating an
expression vector or construct into the genome of a target cell is
to include a `genomic targeting sequence` in the vector or
construct that directs integration of the vector or construct to a
specific genomic site. As an example, the hypoxanthine
phosphoribosyltransferase (HPRT) gene has been used successfully
for this purpose (Bronson, Plaehn et al. 1996; Jasin, Moynahan et
al. 1996). The HPRT gene has additional advantages as a genomic
targeting sequence, for instance its concomitant use as a
selectable marker system. Other genomic targeting sequences that
may be useful in the present invention are described in the art,
for instance (Jasin, Moynahan et al. 1996; van der Weyden, Adams et
al. 2002). The genomic targeting signals as described herein are
useful in certain embodiments of the present invention.
[0032] Introduction of nucleic acids or expression vectors into
cells may be accomplished using techniques well known in the art,
for example microinjection, electroporation, particle bombardment,
or chemical transformation, such as calcium-mediated
transformation, as described for example in Maniatis et al. 1982,
Molecular Cloning, A laboratory Manual, Cold Spring Harbor
Laboratory or in Ausubel et al. 1994, Current protocols in
molecular biology, Jolm Wiley and Sons.
[0033] In certain embodiments of the invention, there are provided
methods of treatment using the nucleic acids or expression vectors,
for instance for gene therapy applications. The nucleic acids or
expression vectors of the present invention may be administered in
isolation, or may be linked to or in combination with tracer
compounds, liposomes, carbohydrate carriers, polymeric carriers or
other agents or excipients as will be apparent to one of skill in
the art. In an alternate embodiment, such compounds may comprise a
medicament, wherein such compounds may be present in a
pharmacologically effective amount.
[0034] The term `medicament` as used herein refers to a composition
that may be administered to a patient or test subject and is
capable of producing an effect in the patient or test subject. The
effect may be chemical, biological or physical, and the patient or
test subject may be human, or a non-human animal, such as a rodent
or transgenic mouse, or a dog, cat, cow, sheep, horse, hamster,
guinea pig, rabbit or pig. The medicament may be comprised of the
effective chemical entity alone or in combination with a
pharmaceutically acceptable excipient.
[0035] The term `pharmaceutically acceptable excipient` may include
any and all solvents, dispersion media, coatings, antibacterial,
antimicrobial or antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
An excipient may be suitable for intravenous, intraperitoneal,
intramuscular, subcutaneous, intrathecal, intraocular, topical or
oral administration. An excipient may include sterile aqueous
solutions or dispersions for extemporaneous preparation of sterile
injectable solutions or dispersion. Use of such media for
preparation of medicaments is known in the art.
[0036] The nucleic acids or expression vectors of the present
invention may be administered to a subject using a viral delivery
system. For instance, the nucleic acids may be inserted into a
viral vector using well known recombinant techniques. The
subsequent viral vector may then be packaged into a virus, such as
adenovirus, lentivirus, attenuated virus, adeno-associated virus
(AAV), and the like. Viral delivery for gene therapy applications
is well known in the art. There exist a variety of options for
viruses suitable for such delivery, which may also involve
selecting an appropriate viral serotype for delivery and expression
in an appropriate tissue.
[0037] Compositions or compounds according to some embodiments of
the invention may be administered in any of a variety of known
routes. Examples of methods that may be suitable for the
administration of a compound include orally, intravenous,
inhalation, intramuscular, subcutaneous, topical, intraperitoneal,
intra-ocular, intra-rectal or intra-vaginal suppository,
sublingual, and the like. The compounds of the present invention
may be administered as a sterile aqueous solution, or may be
administered in a fat-soluble excipient, or in another solution,
suspension, patch, tablet or paste format as is appropriate. A
composition comprising the compounds of the invention may be
formulated for administration by inhalation. For instance, a
compound may be combined with an excipient to allow dispersion in
an aerosol. Examples of inhalation formulations will be known to
those skilled in the art. Other agents may be included in
combination with the compounds of the present invention to aid
uptake or metabolism, or delay dispersion within the host, such as
in a controlled-release formulation. Examples of controlled release
formulations will be known to those of skill in the art, and may
include microencapsulation, embolism within a carbohydrate or
polymer matrix, and the like. Other methods known in the art for
making formulations are found in, for example, "Remington's
Pharmaceutical Sciences", (19th edition), ed. A. Gennaro, 1995,
Mack Publishing Company, Easton, Pa.
[0038] The dosage of the compositions or compounds of some
embodiments of the invention may vary depending on the route of
administration (oral, intravenous, inhalation, or the like) and the
form in which the composition or compound is administered
(solution, controlled release or the like). Determination of
appropriate dosages is within the ability of one of skill in the
art. As used herein, an `effective amount`, a `therapeutically
effective amount`, or a `pharmacologically effective amount` of a
medicament refers to an amount of a medicament present in such a
concentration to result in a therapeutic level of drug delivered
over the term that the drug is used. This may be dependent on mode
of delivery, time period of the dosage, age, weight, general
health, sex and diet of the subject receiving the medicament.
Methods of determining effective amounts are known in the art. It
is understood that it could be potentially beneficial to restrict
delivery of the compounds of the invention to the target tissue or
cell in which protein expression. It is also understood that it may
be desirable to target the compounds of the invention to a desired
tissue or cell type. The compounds of the invention may thus be
coupled to a targeting moiety. The compounds of the invention may
be coupled to a cell uptake moiety. The targeting moiety may also
function as the cell uptake moiety.
UGT8 Mini-Promoters
[0039] The present invention herein provides novel UGT8
mini-promoter sequences which are capable of effecting
transcriptional expression in a spatial and temporal fashion in the
brain and/or eye. Certain UGT8 mini-promoters of the invention
comprise minimal UGT8 promoter elements joined in a non-native
configuration, thus providing advantageous characteristics. Also
provided are novel expression vector compositions comprising UGT8
mini-promoters which allow consistent specific spatiotemporal
transcription of expression sequences. Also provided are novel
methods utilizing these UGT8 mini-promoters and expression
vectors.
[0040] The UGT8 promoters of the invention, as described herein,
are referred to as `mini-promoters` to reflect the fact that the
mini-promoters comprise minimal UGT8 promoter elements sufficient
to drive expression, and that may also be joined by non-native
sequences. In this context, the native intervening sequences may
have been partially or completely removed, and optionally may have
been replaced with non-native sequences. Furthermore, the natural
spatial arrangement of elements may be altered, such that
downstream promoter elements (in natural conformation) are moved
upstream (in non-native conformation). In such a fashion, the
natural spacing of the promoter elements, for instance one or more
human UGT8 regulatory elements corresponding to one or more of SEQ
ID NO: 5-10, and the human UGT8 basal promoter element
corresponding to SEQ ID NO: 3 or 4, or sequences with substantial
functional and/or sequence equivalence, is altered. Additionally,
the orientation of the different promoter elements may be
altered--for instance the regulatory element corresponding to one
or more of SEQ ID NO: 5-10 may be inverted relative to the basal
promoter element corresponding to SEQ ID NO: 3 or 4. An advantage
of such non-native mini-promoters is that the removal of native
intervening sequences reduces the size of the mini-promoter while
maintaining the functional activity of the promoter, thus improving
the utility of the mini-promoter for various applications.
Furthermore, the inversion of an enhancer/promoter element may
allow retention of the enhancer properties without causing
alternate promoter activity.
[0041] The inventors have demonstrated, as illustrated in the
non-limiting Working Examples, that a human UGT8 mini-promoter
having a sequence corresponding to SEQ ID NO: 1 (also referred to
in the Working Examples as Ple240), and which is comprised of 3
human UGT8 regulatory elements (corresponding to SEQ ID NOs: 5, 6
and 7) operably linked in a non-native conformation to a human UGT8
basal promoter having a nucleic acid sequence corresponding to SEQ
ID NO: 3, is capable of directing expression of an expressible
sequence which is operably linked downstream of the UGT8 promoter
in specific cell types in different regions of the brain and/or
eye. The UGT8 regulatory elements (SEQ ID NO: 5-7) and UGT8 basal
promoter element (SEQ ID NO: 3) have sequences which are identical
to those found in the human UGT8 gene. The inventors have further
demonstrated, as illustrated in the non-limiting Working Examples,
that a human UGT8 mini-promoter having a sequence corresponding to
SEQ ID NO: 2 (also referred to in the Working Examples as Ple267),
and which is comprised of 3 human UGT8 regulatory elements
(corresponding to SEQ ID NO: 8-10) operably linked in a non-native
conformation to a human UGT8 basal promoter having a nucleic acid
sequence corresponding to SEQ ID NO: 4, is capable of directing
expression of an expressible sequence which is operably linked
downstream of the UGT8 promoter in specific cell types in different
regions of the brain and/or eye. The UGT8 regulatory elements (SEQ
ID NO: 8-10) and UGT8 basal promoter element (SEQ ID NO: 4) have
sequences which are identical to those found in the human UGT8
gene. It is within the skill of one in the art to locate and
determine these relative positions based on published sequence
information for this gene, for instance found in the GenBank or
PubMed public databases. It is understood that these genomic
coordinates and relative positions are provided for the purposes of
context, and that if any discrepancies exist between published
sequences and the sequence listings provided herein, then the
sequence listings shall prevail.
[0042] Promoters of the present invention may be modified with
respect to the native regulatory and/or native basal promoter
sequence. In general, such modifications will not change the
functional activity of the promoter with respect to cell-type
selectivity; and to the rate of transcription in cells where the
promoter is active. The modified promoter provide for a
transcription rate of an expressible sequence operably linked to a
modified promoter sequence that is at least about 75% the
transcription rate of the promoter sequence of SEQ ID NO: 1 or 2,
at least about 80%, at least about 90%, at least about 95%, at
least about 99%, or more. Methods of assessing promoter strength
and selectivity are known in the art, including, for example,
expression of a reporter sequence in a cell in vivo or in vitro,
and quantitating the reporter activity.
[0043] Modifications of interest include deletion of terminal or
internal regions, and substitution or insertion of residues. The
spacing of conserved sequences may be the same as the native
spacing, or it may be different than the native spacing. The order
of the conserved sequences may be the same as the native order or
the sequences may be rearranged. Sequences set forth in SEQ ID NO:
1 or 2 that are not conserved may be deleted or substituted,
usually modifications that retain the spacing between conserved
sequences is preferred. In general the spacing between the
regulatory element and the basal promoter is not more than about 10
KB, generally not more than about 1 KB, usually not more than about
500 nt, and may be not more than about 100 nt, down to a direct
joining of the two sequences.
[0044] In one embodiment of the invention, there is provided an
isolated nucleic acid fragment comprising a UGT8 mini-promoter,
wherein the UGT8 mini-promoter comprises one or more UGT8
regulatory elements operably linked in a non-native conformation to
a UGT8 basal promoter. The UGT8 mini-promoter may have a nucleic
acid sequence which is substantially similar in sequence and
function to SEQ ID NO: 1 or 2. The UGT8 basal promoter may have a
nucleic acid sequence which is substantially similar in sequence
and function to SEQ ID NO: 3 or 4. The UGT8 regulatory element may
have a nucleic acid sequence which is substantially similar in
sequence and function to one or more of SEQ ID NO: 5-10, e.g.
comprising one, two, three, four or five of the provided regulatory
elements. The UGT8 mini-promoters may further be operably linked to
an expressible sequence, e.g. reporter genes, genes encoding a
polypeptide of interest, regulatory RNA sequences such as miRNA,
siRNA, anti-sense RNA, etc., and the like. Reporter gene sequences
include, for example luciferase, beta-galactosidase, green
fluorescent protein, enhanced green fluorescent protein, and the
like as known in the art. The expressible sequence may encode a
protein of interest, for example a therapeutic protein, receptor,
antibody, growth factor, and the like. The expressible sequence may
encode an RNA interference molecule.
[0045] It is an object of the present invention to provide means of
expressing a gene, protein, RNA interference molecule or the like
in a cell, tissue or organ. As such, the inventors thus provide
novel expression vectors comprising UGT8 mini-promoters which are
capable of accomplishing this task. In one embodiment, there is
provided an expression vector comprising a UGT8 mini-promoter,
wherein the UGT8 mini-promoter comprises one or more UGT8
regulatory element operably linked in a non-native conformation to
an UGT8 basal promoter. The UGT8 mini-promoter may have a nucleic
acid sequence which is substantially similar in sequence and
function to SEQ ID NO: 1 or 2. The UGT8 basal promoter may have a
nucleic acid sequence which is substantially similar in sequence
and function to SEQ ID NO: 3 or 4. The UGT8 regulatory element may
have a nucleic acid sequence which is substantially similar in
sequence and function to one or more of SEQ ID NO: 5-10. The UGT8
mini-promoter may further be operably linked to an expressible
sequence, e.g. reporter genes, genes encoding a polypeptide of
interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense
RNA, etc., and the like. Reporter gene sequences include, for
example luciferase, beta-galactosidase, green fluorescent protein,
enhanced green fluorescent protein, and the like as known in the
art. The expressible sequence may encode a protein of interest, for
example a therapeutic protein, receptor, antibody, growth factor,
and the like. The expressible sequence may encode an RNA
interference molecule. The expression vector may further comprise a
genomic targeting sequence. The genomic targeting sequence may be
HPRT, e.g. human HPRT, mouse HPRT, etc.
[0046] The inventors have herein demonstrated that expression
vectors comprising novel UGT8 mini-promoter elements are capable of
directing transcription of an expression sequence in specific cell
types, for instance in Muller glia cells in the retina (eye) or in
neuronal cells in the brain. In one embodiment of the invention,
there is thus provided a method for expressing a gene, protein, RNA
interference molecule or the like in a cell, the method comprising
introducing into the cell an expression vector comprising an UGT8
mini-promoter element, wherein the UGT8 mini-promoter element
comprises one or more UGT8 regulatory element operably linked in a
non-native conformation to a UGT8 basal promoter element. Cells of
interest include, without limitation, cells of the peripheral or
central nervous system and progenitors thereof, e.g. embryonic stem
cells, neural stem cells, neurons, glial cells, astrocytes,
microgial cells, etc; and/or cells in the eye and progenitors
thereof, e.g. retinal cells, retinal Muller glial cells etc. The
UGT8 mini-promoter may have a nucleic acid sequence which is
substantially similar in sequence and function to SEQ ID NO: 1 or
2. The UGT8 basal promoter may have a nucleic acid sequence which
is substantially similar in sequence and function to SEQ ID NO: 3
or 4. The UGT8 regulatory element may have a nucleic acid sequence
which is substantially similar in sequence and function to one or
more of SEQ ID NO: 5-10. The UGT8 mini-promoter may further be
operably linked to an expressible sequence, e.g. reporter genes,
genes encoding a polypeptide of interest, regulatory RNA sequences
such as miRNA, siRNA, anti-sense RNA, etc., and the like. Reporter
gene sequences include, for example luciferase, beta-galactosidase,
green fluorescent protein, enhanced green fluorescent protein, and
the like as known in the art. The expressible sequence may encode a
protein of interest, for example a therapeutic protein, receptor,
antibody, growth factor, and the like. The expressible sequence may
encode an RNA interference molecule. The expression vector may thus
further comprise a genomic targeting sequence. The genomic
targeting sequence may be HPRT.
[0047] In one embodiment of the invention, there is provided a
method for identifying or labeling a cell, the method comprising
introducing into the cell an expression vector comprising an UGT8
mini-promoter element, wherein the UGT8 mini-promoter element
comprises one or more UGT8 regulatory element operably linked in a
non-native conformation to a UGT8 basal promoter element, and
wherein the expressible sequence comprises a reporter gene. The
UGT8 mini-promoter element may have a nucleic acid sequence
substantially similar in sequence and function to SEQ ID NO: 1 or
2. The UGT8 basal promoter element may have a nucleic acid sequence
substantially similar in sequence and function to SEQ ID NO: 3 or
4. The UGT8 regulatory element may have a nucleic acid sequence
substantially similar in sequence and function to one or more of
SEQ ID NO: 5-10. The inventors have demonstrated that expression
vectors comprising certain human UGT8 promoter elements are capable
of expression in specific regions of the brain and eye, most
notably retinal Muller glial cells in the eye. In some embodiments,
the cell is a peripheral or central nervous system cell or
progenitors thereof, including, without limitation, embryonic stem
cells, neural stem cells, glial cell, neuronal cells, astrocytes,
and the like. In some embodiments, the cell is a cell of the eye
and progenitors thereof, including without limitation retinal
cells, retinal Muller glials cells, and the like. Reporter gene
sequences include, for example luciferase, beta-galactosidase,
green fluorescent protein, enhanced green fluorescent protein, and
the like as known in the art. The expressible sequence may encode a
protein of interest, for example a therapeutic protein, receptor,
antibody, growth factor, RNA interference molecule and the
like.
[0048] In further embodiments of the invention, there is provided a
method for monitoring or tracking the development or maturation of
a cell, the method comprising: 1) introducing into the cell a
expression vector comprising an UGT8 mini-promoter element operably
linked to an expressible sequence, wherein the UGT8 mini-promoter
element comprises one or more UGT8 regulatory elements operably
linked in a non-native conformation to an UGT8 basal promoter
element, and wherein the expressible sequence comprises a reporter
gene; and 2) detecting the expression of the reporter gene in the
cell of in progeny of the cell as a means of determining the
lineage, identity or developmental state of the cell or cell
progeny. The UGT8 mini-promoter element may have a nucleic acid
sequence substantially similar in sequence and function to SEQ ID
NO: 1 or 2. The UGT8 basal promoter element may have a nucleic acid
sequence substantially similar in sequence and function to SEQ ID
NO: 3 or 4. The UGT8 regulatory element may have a nucleic acid
sequence substantially similar in sequence and function to one or
more of SEQ ID NO: 5-10 and/or 6. In such a fashion, one may be
able to follow the development of a parent cell as it
differentiates into more mature cells. As an example, one could
introduce an expression vector comprising the aforementioned UGT8
mini-promoter elements into a pluripotent stem cell, monitor the
expression of the reporter gene that is being expressed by the UGT8
promoter elements during the maturation and differentiation of the
stem cell and thus determine the state of maturation, for instance
in the differentiation of the pluripotent stem cell into a specific
brain or retinal cell type. The inventors have demonstrated that
the UGT8 mini-promoter elements described herein direct
transcriptional expression in certain brain and retinal cell types,
and so detection of reporter gene expression in a cell would thus
be indicative of the cellular identity of the cell as being a
certain type of brain or retinal cell.
[0049] The inventors have herein demonstrated that certain UGT8
mini-promoter elements of the present invention are capable of
driving expression in retinal Muller glial cells. This surprising
expression pattern provides additional methods of use for these
mini-promoter elements. For instance, the UGT8 mini-promoters of
the present invention can be utilized in a gene therapy or cell
therapy application wherein the UGT8 mini-promoters are utilized to
drive expression of a therapeutic or beneficial compound, such as a
protein, in retinal Muller glial cells. In such a way, the
therapeutic or beneficial compound can be useful for a disease or
condition that involves such retinal cells, or which may be
improved by expression of the therapeutic or beneficial compound in
those cells. In certain embodiments of the invention, there is thus
provided a method of treatment of a subject having a disease or
condition of the eye, the method comprising administering to the
subject a therapeutically effective dose of a composition
comprising an UGT8 mini-promoter element, wherein the UGT8
mini-promoter element comprises one or more UGT8 regulatory element
operably linked in a non-native conformation to an UGT8 basal
promoter element. The UGT8 mini-promoter element may have a nucleic
acid sequence substantially similar in sequence and function to SEQ
ID NO: 1 or 2. The UGT8 basal promoter element may have a nucleic
acid sequence substantially similar in sequence and function to SEQ
ID NO: 3 or 4. The UGT8 regulatory element may have a nucleic acid
sequence substantially similar in sequence and function to one or
more of SEQ ID NO: 5-10. The disease or condition may be chosen
from: retinal diseases, retinal degeneration, retinal damage,
blindness, macular degeneration, retinitis pigmentosa, inherited
retinal genetic diseases, diabetic retinopathy, cone rod dystrophy,
hypertensive/diabetic retinopathy. The therapeutic or beneficial
compound may be a light-sensitive compound, for instance rhodopsin,
channel rhodopsin, etc.
[0050] The inventors herein further describe the present invention
by way of the following non-limiting examples:
WORKING EXAMPLES
General Methods
Virus Generation and Analysis
Virus Production
[0051] The Ple240 and Ple267 (UGT8) MiniPromoters were generated by
direct synthesis by DNA2.0 (Menlo Park, Calif., USA) (SEQ ID NO: 1
or 2). Promoter elements were cloned into the pEMS1980 backbone,
containing the iCre reporter, using AvrII and AscI restriction
enzymes. One .mu.g of pEMS1981 plasmid containing either the Ple240
or Ple267 MiniPromoter was prepared by miniprep and sent to the
Vector Core at the University of Pennsylvania (Philadelphia, Pa.,
USA) to be made into AAV9 serotype virus.
Virus Injection
[0052] B6-Gt(ROSA26).sup.tm1Sor females were crossed to
129-Gt(ROSA26).sup.tm1Sor to yield hybrid F1 homozygous pups for
injecting virus. Plug checks were performed on the females such
that the day of birth could be accurately estimated. P0 pups were
used for virus injections. If the female gave birth in the morning,
virus was injected in the afternoon. If she gave birth in the
afternoon, virus was injected the next morning. A standard
injection into the superficial temporal vein of a newborn pup was
performed using 1.times.10.sup.13 GC/mL (genome copies per
milliliter) virus in a total volume of 50 .mu.L (in PBS) with a 30
gauge needle and a 1 cc syringe. After injections, pups were
tattooed for identification and returned to their cage.
Harvesting of Animals
[0053] Virus-injected mice were harvested at P21 or P56 (post-natal
day 21 or 56). Animals were given a lethal dose of avertin injected
intraperitoneally. Thereafter perfusion with 1.times.PBS for 2
minutes and 4% PFA/PBS for 8 minutes was performed. Tissues were
harvested and post-fixed for 1 hour at 4.degree. C. The tissues
were then stored in 0.02% Azide/PBS at 4.degree. C.
Histology
[0054] Tissues were cryoprotected in 30% sucrose/PBS overnight at
4.degree. C. After embedment in OCT the following day, 20 .mu.m
sections were directly mounted onto slides. For X-gal staining,
tissues were rinsed in PBS and Triton-X/PBS and stained in 0.1%
X-gal solution overnight at 30-35.degree. C. After staining
sections were rinsed and counterstained with neutral red,
dehydrated and mounted with coverslips. For co-labeling of X-gal
with markers using immunohistochemistry, standard IHC procedure was
followed and the X-gal stain was performed either prior to primary
antibody incubation or between primary and secondary antibodies,
depending on the strength of the X-gal stain. X-gal stains blue any
cells that have recombined the Gt(ROSA26).sup.tm1Sor locus due to
iCre recombinase activity and thus expressing the
.beta.-galactosidase protein.
Knock-in Mouse Generation and Analysis
Expression Vector
[0055] The nucleic acid fragment corresponding to (SEQ ID NO: 1 or
2) was inserted into the multiple cloning site of pEMS1313 (driving
the lacZ or EGFP reporter) to produce the expression vector (Ple240
or Ple267) used in the experiments.
Derivation of mEMS1202 Embryonic Stem Cells
[0056] Blastocysts were obtained from natural mating of
B6-Hprt.sup.b-m3 homozygous females to 129-ROSA26 heterozygous
males at 3.5 dpc. Blastocysts were flushed from uterine horns as
per (Hogan, Beddington et al. 1994), cultured in EmbryoMax.RTM.
KSOM with 1/2 Amino Acids, Glucose and Phenol Red (Cat # MR-121,
Millipore/Chermicon, Temecula, Calif.) for 3-5 h, and then
transferred onto mitomycin C (mitC; Cat#M4287, Sigma, Oakville, ON)
mitotically inactivated B6-Hprtb-m3, B6129F1, or 129 mouse
embryonic feeders (MEFs) derived from 13.5-day post-coital embryos
(Ponchio, Duma et al. 2000) in 96-well plates containing KSR-ESC
(Knockout.TM. D-MEM, Cat#10829-018, Invitrogen, Burlington, ON)
with 2 mM L-glutamine (Cat#25030-081, Invitrogen, Burlington, ON),
0.1 mM MEM nonessential amino acid solution (Cat#11140-050,
Invitrogen, Burlington, ON) and 16% Knockout.TM. Serum Replacement
(Cat#10828-028, Invitrogen, Burlington, ON)) media (MEF media was
replaced 3-5 hour prior to transfer). Blastocysts were cultured as
per (Cheng, Dutra et al. 2004) with the following modifications:
Cells were cultured for 7-9 days in KSR-ESC with minimal
disturbance (checked on day 2 to determine if the blastocysts had
`hatched` out of the zona pellucida) and no media changes.
Blastocysts which hatched and had a well-developed ICM (inner cell
mass) were treated with 20 .mu.l 0.25% trypsin-EDTA (Invitrogen,
Burlington, ON) for 5 min at 37.degree. C., triturated with a 200
.mu.l Pipetman, inactivated with 30 .mu.l 0.5 mg/ml soybean trypsin
inhibitor (Invitrogen, Burlington, ON), and brought up to 200 .mu.l
with KSR-ESC, then transferred individually to a 24-well MEF plate
containing 1800 .mu.l KSR-ESC, for a total volume of 2 ml.
Beginning 4 days later, KSR-ESC media was replaced with FBS-ESC
media (DMEM (Cat #11960-069, Invitrogen, Burlington, ON) with 2 mM
L-glutamine (Invitrogen, Burlington, ON), 0.1 mM MEM nonessential
amino acid solution (Invitrogen, Burlington, ON), 16% ES Cell
Qualified fetal bovine serum (FBS, Invitrogen, Burlington, ON),
1000 U ESGRO-LIF (Millipore, ESG1107) and 0.01%
.beta.-mercaptoethanol (Sigma, Oakville, ON)) in 25%, 50%, 75%
proportions (respectively) to adapt the cells to FBS-containing
media. On day 7 the cells were trypsinized to one well of a 24 well
plate containing 1 ml of 100% FBS-ESC media, with daily media
replacement. Once confluent, wells containing ESC colonies were
expanded 3.times.24 wells (with MEFs), then passaged to 3.times.24
(with MEFs) and 3.times.12 well (plastic--no MEFs) for DNA
analysis. Once confluent, the 3.times.24 wells were combined,
aliquoted (3 vials), and frozen in ESC-freeze media (50% FBS, 40%
FBS-ESC media, 10% DMSO (Sigma, Oakville, ON), and the 3.times.12
well treated with lysis buffer (Fisher Scientific, Ottawa, ON),
mixed and aliquoted. Cultures were genotyped for X & Y
chromosomes (Clapcote and Roder 2005), Gt(ROSA)26Sortm1Sor and WT
alleles and Hprtb-m3 and WT alleles.
B6129F1-Gt(ROSA)26Sor.sup.tm1Sor/+, Hprt.sup.b-m3/Y (mEMS1204
series) and B6129F1-Gt(ROSA)26Sor.sup.tm1Sor+/+, Hpr.sup.b-m3/Y
(mEMS1202 series) cell lines were identified.
Knock-in at the Hprt Locus
[0057] The expression vector plasmid DNA was purified with Qiagen
Maxi Kit (Qiagen, Mississauga, ON), resuspended in 10:1 Tris-EDTA
(TE, pH7.0) buffer, and linearized with I-Scel (New England
Biolabs, Pickering, ON). Linearized plasmid DNA was resuspended in
85 .mu.l of TE (10:0.1) to a final concentration of 187.5 ng/.mu.l.
Ple240 or Ple267 was targeted in our in-house derived mEMS1202 cell
line. ESCs were grown to confluence on 4-6 T75 flasks of mitC
treated Hprtb-m3 mouse embryonic feeders (MEFs) in FBS-ESC media.
ESCs (1.7-2.5.times.107) in 720 .mu.l 1.times. PBS were added to
the linearized DNA and electroporated in a 4 mm electroporation
cuvette (Bio-Rad Genepulser, Mississauga, ON), at 240 V, 50 .mu.F,
6-10 msec pulse, immediately resuspended in a total volume of 5 ml
of FBS-ESC media and plated onto 5.times.100 mm dishes of mitC
B6129F1 MEFs in a total volume of 12 ml per 100 mm dish. 24-36 h
post-electroporation, correctly targeted homologous recombinants
were selected for using HAT media (FBS-ESC media containing
1.times. HAT ((0.1 mM sodium hypoxanthine, 0.4 mM aminopterin, 0.16
mM thymidine), Cat#21060-017, Invitrogen, Burlington, ON). HAT
media was changed every day for the first 3 days, and then every
3rd day thereafter, for up to 10 days. Individual colonies were
counted and, typically, no more than 2 isolated colonies were
picked per 100 mm dish to optimize for independent homologous
recombination events. These colonies were expanded under standard
protocols for verification of the desired recombination event.
Derivation of Knock-in Mice
[0058] Chimeric mice from targeted ESCs were generated by
microinjection (Hogan, Beddington et al. 1994) into E3.5
blastocysts followed by implantation into the uterine horns of 2.5
day pseudopregnant ICR females. Chimeras were identified and coat
color chimerism determined as outlined below.
[0059] Male chimeras derived from the E14TG2a cell lines were mated
with B6 or B6-Alb females, and germline transmission was identified
in the former case by the transmission of the dominant Aw (white
bellied agouti) allele, making the progeny appear brown with a
cream belly, or in the latter case by the combination of A.sup.w
and Ty.sup.c-ch (chinchilla), making the progeny appear golden.
Non-germline progeny from the cross to B6 were homozygous for the
recessive a (nonagouti) allele and appeared black, whereas
non-germline progeny from the cross to B6-Alb were homozygous for
the recessive Ty.sup.c-2J (albino 2 Jackson) allele and appeared
white.
[0060] Male chimeras derived from the cell lines were mated with
B6-Alb females, and germline transmission identified by the
presence of the dominant Tyr.sup.+ (tyrosinase; wild type) and the
Aw (white bellied agouti) or a (nonagouti) alleles making the
progeny appear brown with a cream belly or black, respectively.
Non-germline progeny were homozygous for the recessive Tyr.sup.c-2J
(albino 2 Jackson) allele and appeared white. All germline female
offspring carry the knock-in X Chromosome and were mated with B6
males.
[0061] N2 offspring were analyzed for the presence of the KI allele
by PCR.
Reporter Gene Detection
[0062] Adult male hemizygous MiniPromoter and age matched control
mice were perfused with 4% paraformaldehyde (PFA) as previously
described (Young, Berry et al. 2002). Whole brains and eyes were
dissected out and post-perfusion immersion fixed with PFA for 2
hours at 4.degree. C. The brains were sectioned using a coronal or
sagittal brain mold (Electron Microscopy Sciences) at 1 mm and
sections were placed in 12-well tissue culture plates. One whole
eye and one half-cut eye, using a razorblade, was also placed in
the plate. LacZ expression was detected by using
5-bromo-4-chloro-3-indolyl-6-D-galactopyranoside (X-Gal) as the
substrate. The X-Gal staining solution contained the following: 1.0
mg/ml X-Gal, 2 mM potassium ferricyanide, 2 mM potassium
ferrocyanide, and 40 mM MgCl.sub.2 in PBS. In brief, brain sections
were rinsed with phosphate buffered saline (PBS), then incubated
with X-Gal (Boeringer Mannheim, Indianapolis, Ind.) at 37.degree.
C., usually overnight. After staining the tissue was rinsed with
PBS and moved into PBS containing 0.02% azide for storage. Eyes
were further processed by post-fixing with 4% PFA for 2 hours at
room temperature. After fixation, eyes were rinsed with PBS and
cryoprotected in 25% sucrose-PBS at 4.degree. C. overnight. Eyes
were removed from the solution and blotted with a KimWipe before
embedment in Optimal Cutting Temperature (OCT) alongside positive
and negative controls. 12 .mu.m sections were taken using a Microm
HM 550 cryostat and directly mounted onto SuperFrost Plus
microscope slides. Bright field images were taken on a Leica MZ125
dissecting microscope and photographed using an Olympus Coolsnap cf
color camera with the ImagePro software package.
Example 1
Selection of UGT8 Mini-Promoter Elements
[0063] The Ple240 construct was designed based on the UGT8 gene. It
combines a basal promoter (SEQ ID NO: 3) along with three upstream
conserved elements with putative regulatory function segment 1 (SEQ
ID NO: 5), segment 2 (SEQ ID NO: 6), and segment 3 (SEQ ID NO: 7).
The Ple267 construct was designed based on the UGT8 gene, and
includes improvements made to the previously described Ple240
design. The construct includes a basal promoter (SEQ ID NO: 4)
along with three upstream conserved elements with putative
regulatory function segment 1 (SEQ ID NO: 8), segment 2 (SEQ ID NO:
9), and segment 3 (SEQ ID NO: 10).
Example 2
Expression of Reporter in Brain and Eye by Ple240 Mini-Promoter
[0064] The Ple240 construct was subsequently tested as a
single-copy site-specific knock-in at the mouse Hprt locus as
previously published (Yang et al. 2009; Portales-Casamar et al.
2010; de Leeuw et al. 2014). Mice were harvested by cardiac
perfusion, sectioned, and stained with the X-gal substrate, which
leaves a blue product after enzymatic cleavage by
beta-galactosidase. In the brain, showed strong fiber-tract
associated staining in all parts of the brain, with cortex layer IV
strongly positive and in thalamic intralaminar and anterior nuclei
groups. The olfactory bulbs have clear glomeruli staining and
ventricles are also lined with positive cells. In the spinal cord,
Ple240 is expressed strongly and predominantly along spinal nerve
cord bundles with weaker expression in the central spinal cord
tissue and in dorsal root ganglia.
[0065] Additionally, the eye showed staining throughout all layers,
which may be indicative of Muller glia cells. Eye expression for
endogenous UGT8 has not been previously reported.
Example 3
Expression of Reporter in Brain by Ple267 Mini-Promoter
[0066] The Ple267 construct was subsequently tested in ssAAV9
driving the icre reporter (Cre recombinase), resulting in vEMS40.
Mice were injected intravenously with virus at post-natal day 0
(P0) (method of (Foust et al. 2009)), and expression analyzed at
P21 and P56 via recombination of the reporter locus
Gt(ROSA26)SortmSor1 (Soriano 1999). Once recombined, this locus
expresses the .beta.-galactosidase (lacZ gene) enzyme. Mice were
harvested by cardiac perfusion, sectioned, and stained with the
X-gal substrate, which leaves a blue product after enzymatic
cleavage by beta-galactosidase. Staining in the brain (e.g. cortex
and corpus callosum) is suggestive of oligodendrocytes. Similarly,
white matter tracts are often positive. The spinal cord has
extensive staining along nerve fiber bundles as well. These results
suggest an oligodendroglial cell type.
LIST OF SEQUENCES
[0067] SEQ ID NO: 1; Ple240 human UGT8 mini-promoter element SEQ ID
NO: 2; Ple267 human UGT8 Mini-Promoter SEQ ID NO: 3; UGT8 Basal
Promoter element #1 SEQ ID NO: 4; UGT8 Basal Promoter element
#2
SEQ ID NO: 5; UGT8 Regulatory Element #1
SEQ ID NO: 6; UGT8 Regulatory Element #2
SEQ ID NO: 7; UGT8 Regulatory Element #3
SEQ ID NO: 8; UGT8 Regulatory Element #4
SEQ ID NO: 9; UGT8 Regulatory Element #5
[0068] SEQ ID NO: 10; UGT8 Regulatory Element #6
Sequence CWU 1
1
1013792DNAH. sapiens 1aaaactgaat acatttcgtc aaaatacaaa agtaaaagta
tttctaggac ttgaaaggtg 60agctaaatga tgagaacaca tggacacata caagtttacc
tatataatga atttgcacat 120gtaccactga acttaaaagt tttttaaaag
tatttctagg aaagtacaaa ctatttaaaa 180tcattagtta gttttgaact
aaaatccttt aaaaatatac aacaaaaaag gtagtttcca 240gatgtccttt
acactgttac tgagaatgta aaatgtcttt cattaatttg gattctttaa
300acattcctct atgatcatat ttaagtgtga aattttactc cctattgttg
tagataatct 360tttagtcagg taatgaatag cacatggtac agaaaaacat
atacatatac attttcattt 420cagcttttct gaagccccat tgtttatttc
aggctgcttt actgagactc cttgcactaa 480acgcctctgg ttgcattcca
ctcaagacaa aggcatattt tagggatgta cctctgacaa 540agaggtattt
atggaccagg aatcagactt tggacaaatg ggcatttcat tataacacag
600cctaattgtt aaagctctta ttgaaaagaa taaaaatttc attagtcatc
acaggaactc 660agcacaaact cctcctcctt ttccccacaa tgcacagaaa
gaatggcagc tttaagctta 720tcattgaaca ttttgaaggc tttggtcctt
ccttcctttt gttccacttg cactaagaag 780tattttagta acaattgtga
aataactgtg gtaataattt taacgaaatt ttataaatgt 840aattgttaaa
cacatctaaa tacacggagc agtcagggag gtgggacgag caggtcattc
900ctcctaacct cacgacatag tcatgctaaa tcttctgctc ctaagtgcag
cacactgggg 960gaaaggaaag gccaaaagag ggagggtcat caactgcaga
gttccacggg gttccaggtg 1020attgcaggtt ataaagcagc ttggtgcctg
tataggctta gtaaatcaag aagtgatgtt 1080aagataatgg aaacagcctg
ctttgtataa atttgtttac tctgttctca ataggtaaca 1140taatttgcat
catatgctct atttatgaca cacctggatt cagaaatgag gtcattaagt
1200tataaacttc atctggttct tttgtgaaaa atagacatca actattagcc
cacttataaa 1260ataacctgta taaattgtac cacctgtctc catgaagcct
cttcaagtgt tcacaaatgt 1320catttcagat gatatttcaa agttatgtat
gtgtttcttc tgtaatatgt caagtttgca 1380atgggttttt attgcaatgt
ttctggttga ttaatgtgag atcaaacaga ccaagtccac 1440agaaataatg
agggaatttt aataccatac tccacattgg ctgaaacttt ctcactgcag
1500gctatgctgt cccattcaac attctataaa ctaaagatga tagggagaat
cttgtttaat 1560ctgaatggga gcttgaagga tacattaaat atggaaaata
tgcatccttc cttaaagaga 1620agtagcaaat gttttcatta ttcatttatt
caaccactta gccaataatg ttaacaatta 1680aaggtgggag cattaaaaaa
aggaaaagtg gggtggggcg gtgaaatgaa cctcactgat 1740ttcttgcttt
tatcatttca aactccaaaa gtgtatttca actattcata tgatgattga
1800ataaacttgt tttacattat ggaacaccct tactgtgtag agctgtgaga
aattcaataa 1860tcagtattta atgtggacaa aaatagatag attagagaaa
caataaacat ggacatatct 1920acacataata tatactcctt ttctatccaa
aaatctgaca aaaaccttac acaatggtca 1980gtttacacca ttttaaggac
aaggatgtgg tgaaacaatt ttaaacattt ttatataata 2040aaagaaaaaa
gttgttttaa ttgctaatga tcaagttttt accaagaatt tacatatgca
2100tcatcaaaca gtagcttgaa actgaagctt gacctttgag aaataaagct
tttaaagtaa 2160atgtctaaat tctcttttct ttattatgtt acatttcatt
tactttaggt tttctagtgc 2220aatttaaata agaaataaat agagagtatg
gtaatcaggt acaaaggctg tgaaaatatt 2280ccctgcattt tgcacaggga
atatgttcaa attcctaatc tgcaaagaag aaaagtgcat 2340tttctttttt
gaaatggcat ttgaagatct ctccatccat gaacattctt gagcatatgg
2400attttacaat aacagcagtg tatattagtt ttttaattta tcattaactg
ataacatata 2460cagtaaaata taatttccgt tttcttatct aagggcagaa
aatcccaacc tatttctgaa 2520tctcactttg gaacaaggac agaatgacct
gacatggcat ttttgatttc caacgtttaa 2580acgcataacg tttgtgtgct
atggaagcat cttagcttct ggaatttatg ctcgacacga 2640aacataaaca
ataaacaatt cagtccacgt tatatagaca tatctcatat atatatgaga
2700tatatatata taaaatatct tacgcacatg cacacttaga ctttctcggt
tttcatgaaa 2760ctcacaatct acctcaggcg ctcaaaggca ctcggcctct
caggtctgag gcaccacaga 2820gaggcttcct tgggcacagt tgcttgctgg
tcaagacgcc aacttggcaa ggttatgctg 2880ctggcaaagg cagattcgtc
agaataaagt cgccacaggc tcaaccaggc aaatcatgaa 2940tggccctttc
agcaggagcc tgagaggagg gatgttattc agcccagcaa ccctatttac
3000tttttttcag gatccccaaa ctcgcgtttt ttaaggcttt ccgctacaag
agagccaaaa 3060ttgaagcctg ggttggcggc agtgcaggta cctgctcacc
taagcatccc tcttttaatt 3120ttcctaactc ctccacccca gagctcactg
agccgccttg atgataaaag gcgggcacag 3180ggactacgtg ggtggtggca
gaaagggcgc gggacacgcc tcgcaaagag ggaagagtgg 3240gcggggccac
gtgccggtgt cagagttcgc aactcgagcg cccagagggc tcgcgaaaag
3300tcccagcctg caagccaacc gcgctcagcg gacgactggc cggatcccaa
cgcgctgccc 3360cttgcccagc ctgcgagcgc gtggtacgaa ggcgcgtctg
catccatgcc ccagcccggg 3420gagctggagg cgctcgcagt cagaggcgag
tgatgctagg ctgagcgcgt ggcggcccgt 3480gtcgtgcccc gctgagccaa
gtgcggaagg gcagcggcgc gctccagctc tgctcgccgc 3540gcgcagggcg
gggggggctg gccgcccgct gggagctgcg gacgagcagg cgcgctgagg
3600acccgaggga ggacacggtt aaagcattgc tatcaactgt gaacccagag
agccctcctt 3660agccaacacg ctaactccga agcctccctt acgcccccga
accaccgaag gcggcgacac 3720ctgattcagc gcacaaacac aggtcccttc
tgtcccggat acaattacgc ggcagacaca 3780cactcaaact cg 379223014DNAH.
sapiens 2aaagtatttc taggaaagta caaactattt aaaatcatta gttagttttg
aactaaaatc 60ctttaaaaat atacaacaaa aaaggtagtt tccagatgtc ctttacactg
ttactgagaa 120tgtaaaatgt ctttcattaa tttggattct ttaaacattc
ctctatgatc atatttaagt 180gtgaaatttt actccctatt gttgtagata
atcttttagt caggtaatga atagcacatg 240gtacagaaaa acatatacat
atacattttc atttcagctt ttctgaagcc ccattgttta 300tttcaggctg
ctttactgag actccttgca ctaaacgcct ctggttgcat tccactcaag
360acaaaggcat attttaggga tgtacctctg acaaagaggt atttatggac
caggaatcag 420actttggaca aatgggcatt tcattataac acagcctaat
tgttaaagct cttattgaaa 480agaataaaaa tttcattagt catcacagga
actcagcaca aactcctcct ccttttcccc 540acaatgcaca gaaagaatgg
cagctttaag cttatcattg aacattttga aggctttggt 600ccttccttcc
ttttgttcca cttgcactaa gaagtatttt agtaacaatt gtgaaataac
660tgtggtaata attttaacga aattttataa atgtatcttc tgctcctaag
tgcagcacac 720tgggggaaag gaaaggccaa aagagggagg gtcatcaact
gcagagttcc acggggttcc 780aggtgattgc aggttataaa gcagcttggt
gcctgtatag gcttagtaaa tcaagaagtg 840atgttaagat aatggaaaca
gcctgctttg tataaatttg tttactctgt tctcaatagg 900taacataatt
tgcatcatat gctctattta tgacacacct ggattcagaa atgaggtcat
960taagttataa acttcatctg gttcttttgt gaaaaataga catcaactat
tagcccactt 1020ataaaataac ctgtataaat tgtaccacct gtctccatga
agcctcttca agtgttcaca 1080acactgattt cttgctttta tcatttcaaa
ctccaaaagt gtatttcaac tattcatatg 1140atgattgaat aaacttgttt
tacattatgg aacaccctta ctgtgtagag ctgtgagaaa 1200ttcaataatc
agtatttaat gtggacaaaa atagatagat tagagaaaca ataaacatgg
1260acatatctac acataatata tactcctttt ctatccaaaa atctgacaaa
aaccttacac 1320aatggtcagt ttacaccatt ttaaggacaa ggatgtggtg
aaacaatttt aaacattttt 1380atataataaa agaaaaaagt tgttttaatt
gctaatgatc aagtttttac caagaattta 1440catatgcatc atcaaacagt
agcttgaaac tgaagcttga cctttgagaa ataaagcttt 1500taaagtaaat
gtctaaattc tcttttcttt attatgttac atttcattta ctttaggttt
1560tctagtgcaa tttaaataag aaataaatag agagtatggt aatcaggtac
aaaggctgtg 1620aaaatattcc ctgcattttg cacagggaat atgttcaaat
tcctaatctg caaagaagaa 1680aagtgcattt tcttttttga aatggcattt
gaagatctct ccatccatga acattcttga 1740gcatatggat tttacaataa
cagcagtgta tattagtttt ttaatttatc attaactgat 1800aacatataca
gtaaaatata atttccgttt tcttatctaa gggcagaaaa tcccaaccta
1860tttctgaatc tcactttgga acaaggacag aatgacctga catggcattt
ttgatttcca 1920acgtttaaac gcataacgtt tgtgtgctat ggaagcatct
tagcttctgg aatttatgct 1980cgacacgaaa cataaacaat aaacaattca
gtccacgtta tatagacata tctcatatat 2040atatgagata tatatatata
aaatatctta cgcacatgca cacttagact ttctcggttt 2100tcatgaaact
cacaatctac ctcaggcgct caaaggcact cggcctctca ggtctgaggc
2160accacagaga ggcttccttg ggcacagttg cttgctggtc aagacgccaa
cttggcaagg 2220ttatgctgct ggcaaaggca gattcgtcag aataaagtcg
ccacaggctc aaccaggcaa 2280atcatgaatg gccctttcag caggagcctg
agaggaggga tgttattcag cccagcaacc 2340ctatttactt tttttcagga
tccccaaact cgcgtttttt aaggctttcc gctacagcac 2400agggactacg
tgggtggtgg cagaaagggc gcgggacacg cctcgcaaag agggaagagt
2460gggcggggcc acgtgccggt gtcagagttc gcaactcgag cgcccagagg
gctcgcgaaa 2520agtcccagcc tgcaagccaa ccgcgctcag cggacgactg
gccggatccc aacgcgctgc 2580cccttgccca gcctgcgagc gcgtggtacg
aaggcgcgtc tgcatccatg ccccagcccg 2640gggagctgga ggcgctcgca
gtcagaggcg agtgatgcta ggctgagcgc gtggcggccc 2700gtgtcgtgcc
ccgctgagcc aagtgcggaa gggcagcggc gcgctccagc tctgctcgcc
2760gcgcgcaggg cggggggggc tggccgcccg ctgggagctg cggacgagca
ggcgcgctga 2820ggacccgagg gaggacacgg ttaaagcatt gctatcaact
gtgaacccag agagccctcc 2880ttagccaaca cgctaactcc gaagcctccc
ttacgccccc gaaccaccga aggcggcgac 2940acctgattca gcgcacaaac
acaggtccct tctgtcccgg atacaattac gcggcagaca 3000cacactcaaa ctcg
30143654DNAH. sapiens 3cagagctcac tgagccgcct tgatgataaa aggcgggcac
agggactacg tgggtggtgg 60cagaaagggc gcgggacacg cctcgcaaag agggaagagt
gggcggggcc acgtgccggt 120gtcagagttc gcaactcgag cgcccagagg
gctcgcgaaa agtcccagcc tgcaagccaa 180ccgcgctcag cggacgactg
gccggatccc aacgcgctgc cccttgccca gcctgcgagc 240gcgtggtacg
aaggcgcgtc tgcatccatg ccccagcccg gggagctgga ggcgctcgca
300gtcagaggcg agtgatgcta ggctgagcgc gtggcggccc gtgtcgtgcc
ccgctgagcc 360aagtgcggaa gggcagcggc gcgctccagc tctgctcgcc
gcgcgcaggg cggggggggc 420tggccgcccg ctgggagctg cggacgagca
ggcgcgctga ggacccgagg gaggacacgg 480ttaaagcatt gctatcaact
gtgaacccag agagccctcc ttagccaaca cgctaactcc 540gaagcctccc
ttacgccccc gaaccaccga aggcggcgac acctgattca gcgcacaaac
600acaggtccct tctgtcccgg atacaattac gcggcagaca cacactcaaa ctcg
6544618DNAH. sapiens 4gcacagggac tacgtgggtg gtggcagaaa gggcgcggga
cacgcctcgc aaagagggaa 60gagtgggcgg ggccacgtgc cggtgtcaga gttcgcaact
cgagcgccca gagggctcgc 120gaaaagtccc agcctgcaag ccaaccgcgc
tcagcggacg actggccgga tcccaacgcg 180ctgccccttg cccagcctgc
gagcgcgtgg tacgaaggcg cgtctgcatc catgccccag 240cccggggagc
tggaggcgct cgcagtcaga ggcgagtgat gctaggctga gcgcgtggcg
300gcccgtgtcg tgccccgctg agccaagtgc ggaagggcag cggcgcgctc
cagctctgct 360cgccgcgcgc agggcggggg gggctggccg cccgctggga
gctgcggacg agcaggcgcg 420ctgaggaccc gagggaggac acggttaaag
cattgctatc aactgtgaac ccagagagcc 480ctccttagcc aacacgctaa
ctccgaagcc tcccttacgc ccccgaacca ccgaaggcgg 540cgacacctga
ttcagcgcac aaacacaggt cccttctgtc ccggatacaa ttacgcggca
600gacacacact caaactcg 6185850DNAH. sapiens 5aaaactgaat acatttcgtc
aaaatacaaa agtaaaagta tttctaggac ttgaaaggtg 60agctaaatga tgagaacaca
tggacacata caagtttacc tatataatga atttgcacat 120gtaccactga
acttaaaagt tttttaaaag tatttctagg aaagtacaaa ctatttaaaa
180tcattagtta gttttgaact aaaatccttt aaaaatatac aacaaaaaag
gtagtttcca 240gatgtccttt acactgttac tgagaatgta aaatgtcttt
cattaatttg gattctttaa 300acattcctct atgatcatat ttaagtgtga
aattttactc cctattgttg tagataatct 360tttagtcagg taatgaatag
cacatggtac agaaaaacat atacatatac attttcattt 420cagcttttct
gaagccccat tgtttatttc aggctgcttt actgagactc cttgcactaa
480acgcctctgg ttgcattcca ctcaagacaa aggcatattt tagggatgta
cctctgacaa 540agaggtattt atggaccagg aatcagactt tggacaaatg
ggcatttcat tataacacag 600cctaattgtt aaagctctta ttgaaaagaa
taaaaatttc attagtcatc acaggaactc 660agcacaaact cctcctcctt
ttccccacaa tgcacagaaa gaatggcagc tttaagctta 720tcattgaaca
ttttgaaggc tttggtcctt ccttcctttt gttccacttg cactaagaag
780tattttagta acaattgtga aataactgtg gtaataattt taacgaaatt
ttataaatgt 840aattgttaaa 8506583DNAH. sapiens 6cacatctaaa
tacacggagc agtcagggag gtgggacgag caggtcattc ctcctaacct 60cacgacatag
tcatgctaaa tcttctgctc ctaagtgcag cacactgggg gaaaggaaag
120gccaaaagag ggagggtcat caactgcaga gttccacggg gttccaggtg
attgcaggtt 180ataaagcagc ttggtgcctg tataggctta gtaaatcaag
aagtgatgtt aagataatgg 240aaacagcctg ctttgtataa atttgtttac
tctgttctca ataggtaaca taatttgcat 300catatgctct atttatgaca
cacctggatt cagaaatgag gtcattaagt tataaacttc 360atctggttct
tttgtgaaaa atagacatca actattagcc cacttataaa ataacctgta
420taaattgtac cacctgtctc catgaagcct cttcaagtgt tcacaaatgt
catttcagat 480gatatttcaa agttatgtat gtgtttcttc tgtaatatgt
caagtttgca atgggttttt 540attgcaatgt ttctggttga ttaatgtgag
atcaaacaga cca 58371705DNAH. sapiens 7agtccacaga aataatgagg
gaattttaat accatactcc acattggctg aaactttctc 60actgcaggct atgctgtccc
attcaacatt ctataaacta aagatgatag ggagaatctt 120gtttaatctg
aatgggagct tgaaggatac attaaatatg gaaaatatgc atccttcctt
180aaagagaagt agcaaatgtt ttcattattc atttattcaa ccacttagcc
aataatgtta 240acaattaaag gtgggagcat taaaaaaagg aaaagtgggg
tggggcggtg aaatgaacct 300cactgatttc ttgcttttat catttcaaac
tccaaaagtg tatttcaact attcatatga 360tgattgaata aacttgtttt
acattatgga acacccttac tgtgtagagc tgtgagaaat 420tcaataatca
gtatttaatg tggacaaaaa tagatagatt agagaaacaa taaacatgga
480catatctaca cataatatat actccttttc tatccaaaaa tctgacaaaa
accttacaca 540atggtcagtt tacaccattt taaggacaag gatgtggtga
aacaatttta aacattttta 600tataataaaa gaaaaaagtt gttttaattg
ctaatgatca agtttttacc aagaatttac 660atatgcatca tcaaacagta
gcttgaaact gaagcttgac ctttgagaaa taaagctttt 720aaagtaaatg
tctaaattct cttttcttta ttatgttaca tttcatttac tttaggtttt
780ctagtgcaat ttaaataaga aataaataga gagtatggta atcaggtaca
aaggctgtga 840aaatattccc tgcattttgc acagggaata tgttcaaatt
cctaatctgc aaagaagaaa 900agtgcatttt cttttttgaa atggcatttg
aagatctctc catccatgaa cattcttgag 960catatggatt ttacaataac
agcagtgtat attagttttt taatttatca ttaactgata 1020acatatacag
taaaatataa tttccgtttt cttatctaag ggcagaaaat cccaacctat
1080ttctgaatct cactttggaa caaggacaga atgacctgac atggcatttt
tgatttccaa 1140cgtttaaacg cataacgttt gtgtgctatg gaagcatctt
agcttctgga atttatgctc 1200gacacgaaac ataaacaata aacaattcag
tccacgttat atagacatat ctcatatata 1260tatgagatat atatatataa
aatatcttac gcacatgcac acttagactt tctcggtttt 1320catgaaactc
acaatctacc tcaggcgctc aaaggcactc ggcctctcag gtctgaggca
1380ccacagagag gcttccttgg gcacagttgc ttgctggtca agacgccaac
ttggcaaggt 1440tatgctgctg gcaaaggcag attcgtcaga ataaagtcgc
cacaggctca accaggcaaa 1500tcatgaatgg ccctttcagc aggagcctga
gaggagggat gttattcagc ccagcaaccc 1560tatttacttt ttttcaggat
ccccaaactc gcgtttttta aggctttccg ctacaagaga 1620gccaaaattg
aagcctgggt tggcggcagt gcaggtacct gctcacctaa gcatccctct
1680tttaattttc ctaactcctc caccc 17058695DNAH. sapiens 8aaagtatttc
taggaaagta caaactattt aaaatcatta gttagttttg aactaaaatc 60ctttaaaaat
atacaacaaa aaaggtagtt tccagatgtc ctttacactg ttactgagaa
120tgtaaaatgt ctttcattaa tttggattct ttaaacattc ctctatgatc
atatttaagt 180gtgaaatttt actccctatt gttgtagata atcttttagt
caggtaatga atagcacatg 240gtacagaaaa acatatacat atacattttc
atttcagctt ttctgaagcc ccattgttta 300tttcaggctg ctttactgag
actccttgca ctaaacgcct ctggttgcat tccactcaag 360acaaaggcat
attttaggga tgtacctctg acaaagaggt atttatggac caggaatcag
420actttggaca aatgggcatt tcattataac acagcctaat tgttaaagct
cttattgaaa 480agaataaaaa tttcattagt catcacagga actcagcaca
aactcctcct ccttttcccc 540acaatgcaca gaaagaatgg cagctttaag
cttatcattg aacattttga aggctttggt 600ccttccttcc ttttgttcca
cttgcactaa gaagtatttt agtaacaatt gtgaaataac 660tgtggtaata
attttaacga aattttataa atgta 6959386DNAH. sapiens 9tcttctgctc
ctaagtgcag cacactgggg gaaaggaaag gccaaaagag ggagggtcat 60caactgcaga
gttccacggg gttccaggtg attgcaggtt ataaagcagc ttggtgcctg
120tataggctta gtaaatcaag aagtgatgtt aagataatgg aaacagcctg
ctttgtataa 180atttgtttac tctgttctca ataggtaaca taatttgcat
catatgctct atttatgaca 240cacctggatt cagaaatgag gtcattaagt
tataaacttc atctggttct tttgtgaaaa 300atagacatca actattagcc
cacttataaa ataacctgta taaattgtac cacctgtctc 360catgaagcct
cttcaagtgt tcacaa 386101315DNAH. sapiens 10cactgatttc ttgcttttat
catttcaaac tccaaaagtg tatttcaact attcatatga 60tgattgaata aacttgtttt
acattatgga acacccttac tgtgtagagc tgtgagaaat 120tcaataatca
gtatttaatg tggacaaaaa tagatagatt agagaaacaa taaacatgga
180catatctaca cataatatat actccttttc tatccaaaaa tctgacaaaa
accttacaca 240atggtcagtt tacaccattt taaggacaag gatgtggtga
aacaatttta aacattttta 300tataataaaa gaaaaaagtt gttttaattg
ctaatgatca agtttttacc aagaatttac 360atatgcatca tcaaacagta
gcttgaaact gaagcttgac ctttgagaaa taaagctttt 420aaagtaaatg
tctaaattct cttttcttta ttatgttaca tttcatttac tttaggtttt
480ctagtgcaat ttaaataaga aataaataga gagtatggta atcaggtaca
aaggctgtga 540aaatattccc tgcattttgc acagggaata tgttcaaatt
cctaatctgc aaagaagaaa 600agtgcatttt cttttttgaa atggcatttg
aagatctctc catccatgaa cattcttgag 660catatggatt ttacaataac
agcagtgtat attagttttt taatttatca ttaactgata 720acatatacag
taaaatataa tttccgtttt cttatctaag ggcagaaaat cccaacctat
780ttctgaatct cactttggaa caaggacaga atgacctgac atggcatttt
tgatttccaa 840cgtttaaacg cataacgttt gtgtgctatg gaagcatctt
agcttctgga atttatgctc 900gacacgaaac ataaacaata aacaattcag
tccacgttat atagacatat ctcatatata 960tatgagatat atatatataa
aatatcttac gcacatgcac acttagactt tctcggtttt 1020catgaaactc
acaatctacc tcaggcgctc aaaggcactc ggcctctcag gtctgaggca
1080ccacagagag gcttccttgg gcacagttgc ttgctggtca agacgccaac
ttggcaaggt 1140tatgctgctg gcaaaggcag attcgtcaga ataaagtcgc
cacaggctca accaggcaaa 1200tcatgaatgg ccctttcagc aggagcctga
gaggagggat gttattcagc ccagcaaccc 1260tatttacttt ttttcaggat
ccccaaactc gcgtttttta aggctttccg ctaca 1315
* * * * *